Qualitative Analysis and Componential Differences of Chemical Constituents in Lysimachiae Herba from Different Habitats (Sichuan Basin) by UFLC-Triple TOF-MS/MS

Lysimachiae Herba (LH), called Jinqiancao in Chinese, is an authentic medical herb in Sichuan Province often used in the prescription of traditional Chinese medicine (TCM). However, in recent years, there has been a lack of comprehensive research on its chemical components. In addition, the landform of Sichuan Province varies greatly from east to west and the terrain is complex and diverse, which has an important influence on the chemical constituents in LH. In this study, ultrafast liquid chromatography coupled with triple-quadrupole time-of-flight tandem mass spectrometry (UFLC-triple TOF-MS/MS) was used to analyze the samples of LH from eight different habitats in Sichuan Basin. The constituents were identified according to the precise molecular weight, the fragment ions of each chromatographic peak and the retention time of the compound obtained by high-resolution mass spectrometry, combined with software database searches, standard comparisons and the related literature. Differential chemical constituents were screened using partial least squares discriminant analysis (PLS-DA) and t-tests. The results showed that a total of 46 constituents were identified and inferred, including flavonoids, phenolic acids, amino acids, tannins, fatty acids and coumarins; the fragmentation pathways of the main constituents were preliminarily deduced. According to the variable importance in projection (VIP) and p-values, four common differential constituents were screened out, 2-O-galloylgalactaric acid, quercetin 3-O-xylosyl-rutinoside, nicotiflorin and kaempferol 3-rutinosyl 7-O-alpha-l-rhamnoside. This study provides basic information for the establishment of a comprehensive quality evaluation system for LH.


Introduction
Lysimachiae Herba (LH), which is the dried whole herb of Lysimachia christinae Hance., has the effect of promoting diuresis and removing jaundice, along with anti-inflammatory and analgesic properties. It is commonly used for symptoms such as jaundice, hypochondriac pain and urolithiasis [1] and it is well known as a key medicine for the treatment of lithiasis. Hitherto, many studies have shown that LH has a variety of pharmacological effects such as the promotion of bile secretion, as well as anti-inflammation, analgesic, bacteriostatic and anti-gout properties [2]. Chemical constituents are the basis for the pharmacological action of traditional Chinese medicine (TCM). Phytochemical analyses have revealed that LH contains multiple chemical constituents, such as flavonoids [3][4][5], phenolic acids [4] and fatty acids [5]. In fact, the quality standard of LH recorded in the Chinese Pharmacopoeia (2020 version) mainly involves the quantification of quercetin and kaempferol [1] and there is still a lack of research on the other constituents of LH. Therefore, it is necessary and important to study its chemical constituents systematically. It is commonly believed that genuine Chinese herbs refer to medicinal materials produced in a specific area of natural conditions. The production is relatively concentrated with certain cultivation techniques and harvesting and processing methods, with excellent quality and effect, as recognized by the clinical practice of TCM. LH is distributed in provinces south of the Yangtze River and Taiwan, but the most well-known habitat is Sichuan Province. Sichuan Province is located inland in southwest China and the upper reaches of the Yangtze River and its terrain is characterized by being high in the west and low in the east. The climate of Sichuan Province is affected by the topography, which can be roughly divided into two climatic zones: Sichuan Plateau and Sichuan Basin [6]. In this study, samples from eight habitats in Sichuan Basin were analyzed to systematically study the chemical constituents of LH and distinguish their differences in different habitats. The research results can preliminarily clarify the material basis of changes in different ecological environments and provide information for rational clinical application.
In recent years, plant metabolomics and liquid chromatography/mass spectrometry (LC-MS) have been widely used in the analysis of complex systems of TCM. Plant metabolomics is a technology for the comprehensive analysis of metabolites in plants, which is especially suitable for multicomponent system analysis of TCM [7]. Combined with the advantages of the high separation of the liquid phase and high sensitivity of mass spectrometry, LC-MS can be used to separate and analyze complex samples and identify their structures. It has been widely used in quantitative and qualitative analysis of complex systems of TCM. Principal component analysis (PCA) is a statistical method that uses the concept of dimension reduction to recombine variables into new composite variables, from which a small number of variables are selected to reflect most of the original information [8]. Partial least squares discriminant analysis (PLS-DA) commonly uses the variable importance in projection (VIP) value to describe the degree of contribution of a variable, which can be considered significant when VIP > 1. Among them, Q 2 > 0. 5 indicates that the predicted value is high, while as R 2 X and R 2 Y approach 1, the model becomes more stable. In this study, a total of 46 chemical constituents in LH were screened out, most of which were flavonoids and phenolic acids. According to the VIP obtained by PLS-DA (VIP > 1) and t-tests (p < 0.05), characteristic peaks of the differentiated chemical constituents were screened out and four commonly differential chemical constituents were finally identified. According to the research ideas and methods of plant metabolomics, this experiment used UFLC-triple TOF-MS/MS to analyze the chemical constituents in LH and combined multivariate statistical analysis to explore the variations in chemical constituents in LH from different areas in Sichuan Basin. Therefore, this study lays a certain foundation for the basic research and quality control of LH medicinal substances.

Optimization of UFLC-Triple TOF-MS/MS Conditions
The effects of methanol-0.1% (v/v) formic acid in water, methanol-water, acetonitrile-0.1% (v/v) formic acid in water and acetonitrile-water as mobile phases under gradient elution on the separation of the peaks in the samples were investigated and we found the methanol-0.1% formic acid in water solution as the mobile phase to achieve a good separation effect. In addition, compared with the electrospray positive ion mode, the negative ion mode was found to have a higher LC/MS response; hence, the samples were measured in negative ion mode in the experiment.

Identification of the Constituents in LH
According to the corresponding chromatographic and mass spectrometric conditions, the chemical constituents of LH samples from eight habitats were identified. The results showed that the constituents identified in the samples from Sichuan Bazhong were more comprehensive. Figure 1 shows the base peak chromatogram (BPC) of the LH sample from Sichuan Bazhong in negative ion mode. A total of 46 constituents were identified, including 25 flavonoids, 11 phenolic acids and 10 other constituents. The detailed information of the identified compounds is shown in Table 1, with their corresponding structures in Figure 2.
Molecules 2022, 27, x FOR PEER REVIEW 3 of 22 methanol at room temperature, a 1:10 solid-liquid ratio, and 60 min ultrasonic extraction were selected as the optimal extraction conditions.

Optimization of UFLC-Triple TOF-MS/MS Conditions
The effects of methanol-0.1% (v/v) formic acid in water, methanol-water, acetonitrile-0.1% (v/v) formic acid in water, and acetonitrile-water as mobile phases under gradient elution on the separation of the peaks in the samples were investigated, and we found the methanol-0.1% formic acid in water solution as the mobile phase to achieve a good separation effect. In addition, compared with the electrospray positive ion mode, the negative ion mode was found to have a higher LC/MS response; hence, the samples were measured in negative ion mode in the experiment.

Identification of the Constituents in LH
According to the corresponding chromatographic and mass spectrometric conditions, the chemical constituents of LH samples from eight habitats were identified. The results showed that the constituents identified in the samples from Sichuan Bazhong were more comprehensive. Figure 1 shows the base peak chromatogram (BPC) of the LH sample from Sichuan Bazhong in negative ion mode. A total of 46 constituents were identified, including 25 flavonoids, 11 phenolic acids, and 10 other constituents. The detailed information of the identified compounds is shown in Table 1, with their corresponding structures in Figure 2.      [30] Note: 1 Comparison with reference standards.    The cleavage pathway is shown in Figure 4D. On the basis of its mode of breakage and comparison with the standard, compound 15 was judged to be chlorogenic acid.

Identification of Amino Acids
Amino acids are a class of organic compounds containing amino and carboxyl groups. In negative ion mode, the secondary mass spectra show fragmentation peaks of [M − H − NH 3 ] − and [M − H − CO 2 ] − , which may be generated by the loss of NH 3 and CO 2 from amino acids. The three amino acids were identified mainly by comparison with the standards, according to the following process: firstly, we used Peakview to find the retention times of the corresponding amino acids in the mixed standard solution; secondly, we compared the m/z of substances with similar retention times (within 0.5 min) in the samples and those with m/z errors of more than 5 ppm were removed; then, the MS/MS patterns of the eligible amino acids consistent with the references and relevant web queries were determined. In the end, compounds 1, 2 and 9 were presumed to be threonine, glutamic acid and phenylalanine, respectively. The comparison of the chromatograms of the three amino-acid standards with those of the extract is shown in Figure 5.
istic quinic acid fragment ion at m/z 191.0596, indicating that the compound often quinic acid (m/z 192) and produce caffeoyl (m/z 161) in the mass spectrometric cleav The cleavage pathway is shown in Figure 4D. On the basis of its mode of breakage comparison with the standard, compound 15 was judged to be chlorogenic acid.

Identification of Amino Acids
Amino acids are a class of organic compounds containing amino and carb groups. In negative ion mode, the secondary mass spectra show fragmentation peak [M − H − NH3] − and [M − H − CO2] − , which may be generated by the loss of NH3 and from amino acids. The three amino acids were identified mainly by comparison with standards, according to the following process: firstly, we used Peakview to find the re tion times of the corresponding amino acids in the mixed standard solution; secondly compared the m/z of substances with similar retention times (within 0.5 min) in the s ples, and those with m/z errors of more than 5 ppm were removed; then, the MS/MS terns of the eligible amino acids consistent with the references and relevant web que were determined. In the end, compounds 1, 2, and 9 were presumed to be threonine, tamic acid, and phenylalanine, respectively. The comparison of the chromatograms o three amino-acid standards with those of the extract is shown in Figure 5.

Identification of Tannins
The basic structure of condensed tannins consists of flavan-3-ols such as (+)-catechin, (−)-epicatechin, or flavan-3,4-diols condensed by C−C at the 4,8-or 4,6-positions. The process of identification was as follows: first, the results of the analysis were imported into Peakview; then, compounds that had a mass error of less than 5 ppm, had the correct isotopic distribution and contained secondary fragments were identified as targets. Combining features of Peakview such as Formula Finder, matching the mass spectrometry data of each chromatographic peak in the database (SciFinder and HMDB) and considering the cleavage law of each peak, compounds 6 and 8 were eventually identified as prodelphinidin B1 and procyanidin B1. Taking  cess of identification was as follows: first, the results of the analysis were imported into Peakview; then, compounds that had a mass error of less than 5 ppm, had the correct isotopic distribution, and contained secondary fragments were identified as targets. Combining features of Peakview such as Formula Finder, matching the mass spectrometry data of each chromatographic peak in the database (SciFinder and HMDB), and considering the cleavage law of each peak, compounds 6 and 8 were eventually identified as prodelphinidin B1 and procyanidin B1. Taking

Identification of Fatty Acids
Fatty acids are a class of carboxylic acid compounds that consist of hydrocarbon groups attached to carboxyl groups. Fatty acids have a better response in negative ion mode. Saturated fatty acids break the C 2 −C 3 bond through γ-hydrogen migration and Mackenzie rearrangement to produce fragments with high intensity, while other fragments are generally (CH 2 ) n −COOH with a 14n difference in relative molecular mass. Reviewing the literature, compounds 3, 4, 5 and 41 were tentatively presumed to be D-mannuronic acid, 2-deoxypentanoic acid, malic acid and 3-methylnonanoic acid. Taking  Fatty acids are a class of carboxylic acid compounds that consist of hydrocarbon groups attached to carboxyl groups. Fatty acids have a better response in negative ion mode. Saturated fatty acids break the C2−C3 bond through γ-hydrogen migration and Mackenzie rearrangement to produce fragments with high intensity, while other fragments are generally (CH2)n−COOH with a 14n difference in relative molecular mass. Reviewing the literature, compounds 3, 4, 5, and 41 were tentatively presumed to be D-mannuronic acid, 2-deoxypentanoic acid, malic acid, and 3-methylnonanoic acid. Taking

Identification of Coumarins
Coumarins have a lactone structure and can be structurally viewed as cis-o-hydroxycinnamates formed by intramolecular dehydration of the ring. Oxygen-containing functional groups can be substituted at various positions on the benzene ring, commonly including hydroxyl, methoxy, and sugar groups. Compound 18 had fragments at m/z 177.0221 [M − H] − in negative mode, with a major secondary fragment at m/z 149.0267 reduced by 28 from m/z 177.0221, presumably caused by the loss of CO, which is characteristic of the cleavage of coumarins. Therefore, it could be presumed that compound 18 was 6,7-dihydroxycoumarin, and its MS 2 spectrum and speculated fragmentation pathways are shown in Figure 8.

Identification of Coumarins
Coumarins have a lactone structure and can be structurally viewed as cis-o-hydroxycinnamates formed by intramolecular dehydration of the ring. Oxygen-containing functional groups can be substituted at various positions on the benzene ring, commonly including hydroxyl, methoxy and sugar groups. Compound 18 had fragments at m/z 177.0221 [M − H] − in negative mode, with a major secondary fragment at m/z 149.0267 reduced by 28 from m/z 177.0221, presumably caused by the loss of CO, which is characteristic of the cleavage of coumarins. Therefore, it could be presumed that compound 18 was 6,7-dihydroxycoumarin and its MS 2 spectrum and speculated fragmentation pathways are shown in Figure 8.

PCA of the Samples
PCA was used to analyze the differences between the samples of LH from different habitats (S1, Bazhong; S2, Guangyuan; S3, Yibin; S4, Zigong; S5, Deyang; S6, Suining; S7, Leshan; S8, Nanchong) and the correlation between samples. The PCA model parameters of R 2 X = 0.865 and Q 2 = 0.785 showed that the models were effective and reliable. As shown in Figure 9, the samples of LH from the eight habitats were clustered into one category, and the distribution results were relatively ideal. Among them, the samples of S4 and S7 were highly aggregated and had little difference. The relative dispersion of S3 and other habitats indicated that the chemical constituents of S3 differed significantly from the samples of other habitats.

PCA of the Samples
PCA was used to analyze the differences between the samples of LH from different habitats (S1, Bazhong; S2, Guangyuan; S3, Yibin; S4, Zigong; S5, Deyang; S6, Suining; S7, Leshan; S8, Nanchong) and the correlation between samples. The PCA model parameters of R 2 X = 0.865 and Q 2 = 0.785 showed that the models were effective and reliable. As shown in Figure 9, the samples of LH from the eight habitats were clustered into one category and the distribution results were relatively ideal. Among them, the samples of S4 and S7 were highly aggregated and had little difference. The relative dispersion of S3 and other habitats indicated that the chemical constituents of S3 differed significantly from the samples of other habitats.

PCA of the Samples
PCA was used to analyze the differences between the samples of LH from different habitats (S1, Bazhong; S2, Guangyuan; S3, Yibin; S4, Zigong; S5, Deyang; S6, Suining; S7, Leshan; S8, Nanchong) and the correlation between samples. The PCA model parameters of R 2 X = 0.865 and Q 2 = 0.785 showed that the models were effective and reliable. As shown in Figure 9, the samples of LH from the eight habitats were clustered into one category, and the distribution results were relatively ideal. Among them, the samples of S4 and S7 were highly aggregated and had little difference. The relative dispersion of S3 and other habitats indicated that the chemical constituents of S3 differed significantly from the samples of other habitats. Figure 9. PCA score plot of LH samples from different habitats. Figure 9. PCA score plot of LH samples from different habitats.

PLS-DA of the Samples
In this experiment, the samples from the other seven habitats were compared with the samples from Bazhong and analyzed by PLS-DA. The results are shown in Figure 10a. As can be seen from the Figure 10, the samples from each habitat were obviously separated from S1 samples along the PC1 axis. The models were tested with 200 permutations and the results are listed in Table 2. The results showed that the models did not overfit, indicating that they were effective and reliable. According to the VIP score chart (Figure 10b) and t-tests corresponding to the model, differential chemical constituents (VIP > 1) of samples from different habitats in Sichuan Basin were screened out and the number of characteristic peaks is shown in Table 2.

PLS-DA of the Samples
In this experiment, the samples from the other seven habitats were compared with the samples from Bazhong and analyzed by PLS-DA. The results are shown in Figure 10a. As can be seen from the Figure 10, the samples from each habitat were obviously separated from S1 samples along the PC1 axis. The models were tested with 200 permutations, and the results are listed in Table 2. The results showed that the models did not overfit, indicating that they were effective and reliable. According to the VIP score chart ( Figure  10b) and t-tests corresponding to the model, differential chemical constituents (VIP > 1) of samples from different habitats in Sichuan Basin were screened out, and the number of characteristic peaks is shown in Table 2.

Identification of the Differential Chemical Constituents
A total of four common differential chemical constituents, 2-O-Galloylgalactaric acid, quercetin 3-O-xylosyl-rutinoside, kaempferol 3-O-rutinoside and kaempferol 3-rutinoside 7-rhamnoside, were identified from the eight samples of LH from different habitats in Sichuan Basin. The peak area of each common differential constituent was used as its relative content. The average value and standard deviation of the peak area of the same chemical constituent in different samples were calculated to obtain the relative content changes of common differential constituents between different samples ( Figure 11). As shown in the figure, the content of all four differential constituents of LH from S1 was high.
in Sichuan Basin. The peak area of each common differential constituent was used as its relative content. The average value and standard deviation of the peak area of the same chemical constituent in different samples were calculated to obtain the relative content changes of common differential constituents between different samples ( Figure 11). As shown in the figure, the content of all four differential constituents of LH from S1 was high. Figure 11. Relative contents of the common differential chemical constituents.

Discussion
At present, there are few studies on the components of LH. In the current national standard, the quality control indicator of LH is the content of two glycosidic constituents, quercetin and kaempferol, which have small polarity. This study used UFLC-triple TOF-MS/MS to comprehensively analyze the chemical constituents of LH. Finally, 46 chemical constituents in LH were identified (Table 1), including flavonoids, phenolic acids, and amino acids. Generally speaking, the accumulation of active ingredients in LH varies greatly due to the different ecological environments. Accordingly, the quality of herbs also varies, which makes it difficult to standardize commercial herbs and ensure their effectiveness for clinical use. Therefore, it is of great importance to study the constituents of LH from different habitats in Sichuan Province. This experiment focused on the compositional analysis of herbs from eight different habitats (Bazhong, Guangyuan, Yibin, Zigong, Deyang, Suining, Leshan, and Nanchong) in Sichuan Basin. The PCA showed that the samples from the eight different habitats in Sichuan basin were clustered into one category, and the distribution results were relatively ideal. According to the results of PLS-DA and VIP, four common differential chemical constituents were screened out from samples in Sichuan basin. The content of all four differential constituents in the samples of LH in Sichuan Bazhong was high, and the overall level was good; the content of all four differential constituents in the sample of LH from Sichuan Yibin was low. However, the influence of different habitats on the quality of LH is still unclear in many aspects. Therefore, it is necessary to analyze the LH constituents of the Sichuan Plateau and to further develop a comparison of all samples from Sichuan. The results provide basic data for revealing the Figure 11. Relative contents of the common differential chemical constituents.

Discussion
At present, there are few studies on the components of LH. In the current national standard, the quality control indicator of LH is the content of two glycosidic constituents, quercetin and kaempferol, which have small polarity. This study used UFLC-triple TOF-MS/MS to comprehensively analyze the chemical constituents of LH. Finally, 46 chemical constituents in LH were identified (Table 1), including flavonoids, phenolic acids and amino acids. Generally speaking, the accumulation of active ingredients in LH varies greatly due to the different ecological environments. Accordingly, the quality of herbs also varies, which makes it difficult to standardize commercial herbs and ensure their effectiveness for clinical use. Therefore, it is of great importance to study the constituents of LH from different habitats in Sichuan Province. This experiment focused on the compositional analysis of herbs from eight different habitats (Bazhong, Guangyuan, Yibin, Zigong, Deyang, Suining, Leshan and Nanchong) in Sichuan Basin. The PCA showed that the samples from the eight different habitats in Sichuan basin were clustered into one category and the distribution results were relatively ideal. According to the results of PLS-DA and VIP, four common differential chemical constituents were screened out from samples in Sichuan basin. The content of all four differential constituents in the samples of LH in Sichuan Bazhong was high and the overall level was good; the content of all four differential constituents in the sample of LH from Sichuan Yibin was low. However, the influence of different habitats on the quality of LH is still unclear in many aspects. Therefore, it is necessary to analyze the LH constituents of the Sichuan Plateau and to further develop a comparison of all samples from Sichuan. The results provide basic data for revealing the influence of the ecological environment on the synthesis and accumulation of metabolites of LH, as well as the quality formation mechanism of the herb.

Plant Materials
The samples of LH were collected in the field in Sichuan Province. The materials were identified by Professor Xunhong Liu (Department for Authentication of Chinese Medicines, School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, China) as the dried whole herb of Lysimachia christinae Hance. Voucher specimens were deposited in the laboratory of Chinese medicine identification, Nanjing University of Chinese Medicine. The source information of the LH samples is shown in Table 3.

. Preparation of Standard and Sample Solutions
The above 23 standards were weighed in a 5 mL volumetric flask using a 1/1,000,000 electronic analytical balance (ME36S, Sydos, Germany), dissolved in methanol and prepared into the corresponding concentrations of standard solutions. Then, 50 µL of the above standard solutions was placed into a 10 mL volumetric flask and methanol was added to prepare a mixed standard solution. The concentration of the mixed standard solution was 5 µg/mL. All solutions were stored at 4 • C for further analysis.
The samples were crushed and passed through a No. 3 sieve and then the powder was dried to constant weight. Next, 0.5 g of dried powder was accurately weighed into a 50 mL centrifuge tube and ultrasonically extracted with 5 mL of 80% methanol for 60 min at room temperature. After extraction for a few minutes, the weight was made up with 80% methanol. The supernatant was taken and centrifuged at 13,000 rpm for 10 min before filtering through a 0.22 µm membrane (Jinteng laboratory equipment Co., Ltd., Tianjin, China) prior to UFLC-triple TOF-MS/MS analysis.

Analysis of the Differential Constituents in LH from Different Habitats
Markerview 1.2.1 software (Sciex AB, Framinghan, MA, USA) was used to perform peak matching, peak alignment and noise filtering for the raw mass spectrometry data. The results were further imported into SIMCA-P 13.0 software (Umetrics AB, Umea, Sweden). On the basis of the above qualitative results, PCA and PLS-DA were used to perform dimensionality reduction analysis on the data to obtain information about differences between groups. The characteristic peaks of the differential chemical components were screened according to the VIP (VIP > 1) and t-test (p < 0.05) results obtained from PLS-DA.

Conclusions
In this study, UFLC-triple TOF-MS/MS was used to analyze the components from eight different habitats in Sichuan basin. According to relevant mass spectrometry data, reference materials and the literature, 46 chemical constituents were identified. The fragmentation pathways of flavonoids, phenolic acids, tannins, amino acids, fatty acids and coumarins were preliminarily deduced by the fragmentation behavior of the major components. PCA, PLS-DA and t-tests were used to identify the different common chemical components of LH from different habitats and their different contents were compared at the same time. Finally, we found that LH from Sichuan Bazhong was best among the eight habitats. In conclusion, these results can help us to better understand the chemical constituents and componential differences of chemical constituents in LH from different habitats, as well as provide data for further exploring the functional material basis and clinical application of LH.

Data Availability Statement:
The data presented in this study are available within the article.

Conflicts of Interest:
The authors declare no conflict of interest.
Sample Availability: Samples of the Lysimachiae Herba are available from the authors.